Differential Expression of the Diverse Plant Actins

描述（由申请者提供）：我们提出有两类基于肌动蛋白的细胞骨架系统，营养（VEG）和生殖（REP），它们在维管植物中以相对独立的方式运作和进化了超过3.5亿年，类似于脊椎动物的细胞质和肌肉肌动蛋白系统。为了了解基因调控和蛋白异变序列差异的相对重要性，我们研究了编码8种肌动蛋白（ACT）、5种profins （PRF）、11种肌动蛋白解聚因子（ADF）和6种核肌动蛋白相关蛋白（ARP）的基因家族。ACT、ADF和PRF细胞骨架家族包含具有vegf或REP表达模式的古老基因亚类，而ARPs则是组成型表达。敲除ACT、ADF、PRF和ARP基因中的等位基因揭示了在多细胞发育的每个阶段存在大量多样的表型缺陷。我们构建了具有单一VEG肌动蛋白同变异体的高度合成的健康植物，以证明VEG基因调控的重要性。我们能够用REP抑制异位REP肌动蛋白基因的表达，但不能抑制VEG， ADFs和PRFs，这表明了等变异体特异性相互作用的重要性。虽然ACT、ADF和PRF缺陷导致细胞骨架的改变，但一些等位基因在转录因子基因表达上共享表观遗传变化，导致ARP突变体发育改变。由于已知核ARPs主要在含有常规肌动蛋白亚基的染色质重塑复合体中起作用，这些结果导致了一种新的建议：发育主调控因子的转录表达由在染色质重塑复合体中起作用的常规肌动蛋白控制，而细胞核中肌动蛋白的水平由adf和prf控制。我们下一个资助期的具体目标是：1)表征ACT、ADF和PRF突变体的表型；2)描述基因和同型变异特异性相互作用及其在多细胞发育中的作用；3)探讨这些蛋白在核表观遗传和细胞质细胞骨架功能之间的平衡作用。蛋白质同工变异体之间的差异及其差异调控在动物多细胞发育中起着重要作用。毫无疑问，我们的实验设计和研究成果为分子医学（例如，人类线状肌动蛋白突变）的此类研究勾勒了一条路径。

DESCRIPTION (provided by applicant): We propose there are two classes of actin-based cytoskeletal systems, vegetative (VEG) and reproductive (REP), which have functioned and evolved with relative independence for more than 350 million years in vascular plants, similar to the cytoplasmic and muscle actin systems in vertebrates. To understand the relative importance of differences in gene regulation and protein isovariant sequence, we examined the gene families encoding eight actins (ACT), five profilins (PRF), eleven actin depolymerizing factors (ADF), and six nuclear actin-related proteins (ARP). The ACT, ADF, and PRF cytoskeletal families contained ancient subclasses of genes with VEG or REP expression patterns, while the ARPs were constitutively expressed. Knockout and knockdown alleles among the ACT, ADF, PRF, and ARP genes revealed numerous and diverse phenotypic defects in every stage of multicellular development. We constructed highly synthetic healthy plants with a single VEG actin isovariant to demonstrate the importance of VEG gene regulation. We were able to suppress ectopic REP actin gene expression with REP, but not VEG, ADFs and PRFs demonstrating the importance of isovariant-specific interactions. While ACT, ADF, and PRF defects resulted in alterations in the cytoskeleton, a few alleles shared epigenetic changes in transcription factor gene expression leading to altered development with ARP mutants. Because nuclear ARPs are known to function primarily in chromatin remodeling complexes containing conventional actin subunits, these results led to a new proposal: the transcriptional expression of master regulators of development is controlled by conventional actins functioning in chromatin remodeling complexes, with the level of actin in the nucleus controlled by ADFs and PRFs. Our Specific Aims in the next grant period are: 1) to characterize the phenotypes of ACT, ADF, and PRF mutants; 2) to characterize gene and isovariant specific interactions and their roles in multicellular development; and 3) to explore how the roles of these proteins are balanced between nuclear epigenetic and cytoplasmic cytoskeletal functions. Differences among protein isovariants and their differential regulation are now widely recognized to play an essential role in animal multicellular development. Undoubtedly, our experimental designs and research outcomes outline a path for such studies in molecular medicine (e.g., human nemaline actin mutations).